Figure 2: HDAC5 and LSD1 physically interact in breast cancer cells. (a) MDA-MB-231 or MCF10A–CA1a cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5 plasmids. Immunoprecipitation (IP) was performed with anti-LSD1 antibody followed by immunoblotting (IB) with anti-LSD1, anti-FLAG or anti-HDAC5 antibodies, respectively. (b) Whole cell lysates were immunoprecipitated with anti-LSD1 antibody followed by IB with anti-HDAC5 and LSD1 antibodies in indicated breast cancer cell lines. IgG was used as negative control. (c) MDA-MB-231 cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5-FLAG plasmids, and IP was performed with anti-FLAG followed by IB with anti-LSD1 and anti-FLAG antibodies, respectively. (d) Schematic representation of full length and deletion mutants of HDAC5-FLAG constructs. (e) FLAG-tagged full-length or deletion mutants of HDAC5 were expressed in MDA-MB-231 cells. Extracts were immunoprecipitated with anti-FLAG antibody, and bound LSD1 was examined by IB using anti-LSD1 antibody. IB with anti-FLAG was used to detect the levels of FLAG-tagged HDAC5 full-length or deletion mutants in IP and input samples (10%). (f) MDA-MB-231 cells were transfected with plasmids expressing FLAG-tagged full-length or deletion mutants of HDAC5 proteins. Immunofluorescence study was performed using anti-FLAG antibody. DAPI was used as a control for nuclear staining. All the experiments were performed three times with similar results.

Mentions:
To address whether LSD1 and HDAC5 physically interact, a co-immunoprecipitation study was carried out in MDA-MB-231 and MCF10A–CA1a cells transiently transfected with pcDNA3.1 or pcDNA3.1-FLAG-HDAC5 plasmids. After immunoprecipitation (IP) with LSD1 antibody, we found that both endogenous and exogenous HDAC5 proteins were co-immunoprecipitated with LSD1 protein (Figure 2a). The interaction between native LSD1 and HDAC5 was further validated in additional breast cancer cell lines (Figure 2b). A similar result was obtained in the reciprocal immunoprecipitation using anti-FLAG antibody to confirm that LSD1 was co-immunoprecipitated with FLAG-HDAC5 (Figure 2c). To precisely map the HDAC5 domain(s) responsible for interaction with LSD1, we expressed a series of HDAC5 deletion mutants engineered in pcDNA3.1-FLAG plasmids in MDA-MB-231 cells (Figure 2d). Immunoprecipitation assays of cells transfected with full length HDAC5 cDNA confirmed the HDAC5-LSD1 interaction. Deletion of an N-terminal myocyte enhancer factor-2 (MEF2) binding domain (HDAC5-Δ1) alone had no impact on HDAC5-LSD1 interaction. However, removal of both the MEF2 domain and nuclear localization sequence (NLS) (HDAC5-Δ2) completely abolished HDAC5-LSD1 interaction. Further deletion of an N-terminal HDAC and nuclear export sequence (NES) (HDAC5-Δ3) and MEF2 domain (HDAC5-Δ4) did not adversely alter LSD1 binding with HDAC5 fragments (Figure 2e). Immunofluorescence studies showed nuclear localization of full length HDAC5, HDAC5-Δ1, HDAC5-Δ3 and HDAC5-Δ4. Depletion of the NLS-containing domain (HDAC5-Δ2) completely blocked HDAC5 nuclear translocation (Figure 2f). In vitro pull-down assays by using His-tag recombinant LSD1 protein incubating with HDAC5 full length or deletion mutants validated that HDAC5 domain containing NLS element is essential for interaction with LSD1 (Supplementary Figure 4).

Figure 2: HDAC5 and LSD1 physically interact in breast cancer cells. (a) MDA-MB-231 or MCF10A–CA1a cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5 plasmids. Immunoprecipitation (IP) was performed with anti-LSD1 antibody followed by immunoblotting (IB) with anti-LSD1, anti-FLAG or anti-HDAC5 antibodies, respectively. (b) Whole cell lysates were immunoprecipitated with anti-LSD1 antibody followed by IB with anti-HDAC5 and LSD1 antibodies in indicated breast cancer cell lines. IgG was used as negative control. (c) MDA-MB-231 cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5-FLAG plasmids, and IP was performed with anti-FLAG followed by IB with anti-LSD1 and anti-FLAG antibodies, respectively. (d) Schematic representation of full length and deletion mutants of HDAC5-FLAG constructs. (e) FLAG-tagged full-length or deletion mutants of HDAC5 were expressed in MDA-MB-231 cells. Extracts were immunoprecipitated with anti-FLAG antibody, and bound LSD1 was examined by IB using anti-LSD1 antibody. IB with anti-FLAG was used to detect the levels of FLAG-tagged HDAC5 full-length or deletion mutants in IP and input samples (10%). (f) MDA-MB-231 cells were transfected with plasmids expressing FLAG-tagged full-length or deletion mutants of HDAC5 proteins. Immunofluorescence study was performed using anti-FLAG antibody. DAPI was used as a control for nuclear staining. All the experiments were performed three times with similar results.

Mentions:
To address whether LSD1 and HDAC5 physically interact, a co-immunoprecipitation study was carried out in MDA-MB-231 and MCF10A–CA1a cells transiently transfected with pcDNA3.1 or pcDNA3.1-FLAG-HDAC5 plasmids. After immunoprecipitation (IP) with LSD1 antibody, we found that both endogenous and exogenous HDAC5 proteins were co-immunoprecipitated with LSD1 protein (Figure 2a). The interaction between native LSD1 and HDAC5 was further validated in additional breast cancer cell lines (Figure 2b). A similar result was obtained in the reciprocal immunoprecipitation using anti-FLAG antibody to confirm that LSD1 was co-immunoprecipitated with FLAG-HDAC5 (Figure 2c). To precisely map the HDAC5 domain(s) responsible for interaction with LSD1, we expressed a series of HDAC5 deletion mutants engineered in pcDNA3.1-FLAG plasmids in MDA-MB-231 cells (Figure 2d). Immunoprecipitation assays of cells transfected with full length HDAC5 cDNA confirmed the HDAC5-LSD1 interaction. Deletion of an N-terminal myocyte enhancer factor-2 (MEF2) binding domain (HDAC5-Δ1) alone had no impact on HDAC5-LSD1 interaction. However, removal of both the MEF2 domain and nuclear localization sequence (NLS) (HDAC5-Δ2) completely abolished HDAC5-LSD1 interaction. Further deletion of an N-terminal HDAC and nuclear export sequence (NES) (HDAC5-Δ3) and MEF2 domain (HDAC5-Δ4) did not adversely alter LSD1 binding with HDAC5 fragments (Figure 2e). Immunofluorescence studies showed nuclear localization of full length HDAC5, HDAC5-Δ1, HDAC5-Δ3 and HDAC5-Δ4. Depletion of the NLS-containing domain (HDAC5-Δ2) completely blocked HDAC5 nuclear translocation (Figure 2f). In vitro pull-down assays by using His-tag recombinant LSD1 protein incubating with HDAC5 full length or deletion mutants validated that HDAC5 domain containing NLS element is essential for interaction with LSD1 (Supplementary Figure 4).